Design, synthesis and perception of fluorescently labeled isoprenoid cytokinins

Kubiasová, Karolina; Mik, Václav; Nisler, Jaroslav; Hönig, Martin; Husičková, Alexandra; Spíchal, Lukáš; Pěkná, Zuzana; Šamajová, Olga; Doležal, Karel; Plíhal, Ondřej; Benková, Eva; Strnad, Miroslav; Plíhalová, Lucie

doi:10.1016/j.phytochem.2018.02.015

Cited by 10 publications

(7 citation statements)

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“…Cell-specific sorting has been employed to gain more accurate insight into auxin [ 33 , 185 ] and CK [ 186 ] distributions in Arabidopsis root tips, and flow cytometric techniques for the sorting of organelles may soon provide a better view on subcellular distributions. Another possible approach for visualizing auxin and CK distributions at the cellular or subcellular level is using novel synthetic analogues labelled with 7-nitro-2,1,3-benzoxadiazole (NBD), for example, that have been recently developed to mimic native phytohormones in vivo [ 187 , 188 , 189 ]. A combination of all these methodologies with the use of mathematical modelling [ 78 , 122 , 190 ] to parameterize auxin homeostasis at cellular and subcellular levels will undoubtedly lead to far more detailed insights into the secrets of plant developmental control.…”

Section: Future Perspectivesmentioning

confidence: 99%

Auxins and Cytokinins—The Role of Subcellular Organization on Homeostasis

Skalický

Kubeš

Napier

et al. 2018

IJMS

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Plant hormones are master regulators of plant growth and development. Better knowledge of their spatial signaling and homeostasis (transport and metabolism) on the lowest structural levels (cellular and subcellular) is therefore crucial to a better understanding of developmental processes in plants. Recent progress in phytohormone analysis at the cellular and subcellular levels has greatly improved the effectiveness of isolation protocols and the sensitivity of analytical methods. This review is mainly focused on homeostasis of two plant hormone groups, auxins and cytokinins. It will summarize and discuss their tissue- and cell-type specific distributions at the cellular and subcellular levels.

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Section: Future Perspectivesmentioning

confidence: 99%

Auxins and Cytokinins—The Role of Subcellular Organization on Homeostasis

Skalický

Kubeš

Napier

et al. 2018

IJMS

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“…S6). Two carbon linker terminated with amino group suitable for consecutive NBD fluorophore attachment was coupled to the purine N 9 position by the reaction of 6-chloropurine with N -Boc-ethanolamine under Mitsunobu conditions 7 . Later, nucleophilic substitution of C6 chlorine with appropriate amines in boiling alcohols followed by Boc protective group cleavage provided purine intermediates for fluorescent labelling.…”

Section: Supplementary Methodsmentioning

confidence: 99%

“…4-chloro-7-nitro-1,2,3-benzoxadiazol was used as NBD donor and was linked to the purine primary amino group in MeOH under basic conditions. Physico-chemical characterization of the synthesized compounds was done according to 7 .…”

Section: Supplementary Methodsmentioning

confidence: 99%

“…Synthesized according to 7 Redish-brown solid, yield 68 %, HPLC purity 99.9, ESI + -MS 342 (100, [M+H] + ), 1 H-NMR (500 MHz, DMSO- d 6 ) δ (ppm): 3.92 (s, 2H, CH 2 C H 2 NHNBD), 4.46 (s, 2H, C H 2 CH 2 NHNBD), 6.44 (d, J = 8.3 Hz, 1H, NBD H6), 7.19 (s, 2H, NH 2 ), 8.04 (s, 1H, pur C2), 8.09 (s, 1H, pur H8), 8.46 (d, J = 8.9 Hz, 1H, NBD H5), 9.48 (s, 1H, CH 2 CH 2 N H NBD). 13 C-NMR (125 MHz, DMSO- d 6 ) δ (ppm): 41.3 ( C H 2 CH 2 NHNBD), 42.8 (CH 2 C H 2 NHNBD), 99.3 (NBD C6), 118.7 (pur C5), 121.3 (NBD), 137.7 (NBD C5), 141.0 (pur C8), 144.0 (NBD), 144.4 (NBD), 144.9 (NBD), 149.7 (pur C4), 152.3 (pur C2), 155.9 (pur C6).…”

Section: Supplementary Methodsmentioning

confidence: 99%

“…However, localization of cytokinin HK receptors to the PM, although initially suggested 6 , remains ambiguous. Here, by monitoring subcellular localizations of the fluorescently labelled natural cytokinin probe iP-NBD 7 and the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin fluoroprobes and receptors can enter the secretory pathway and reach the PM. We demonstrate that in cells of the root apical meristem, CRE1/AHK4 localizes to the PM and the cell plate of dividing meristematic cells.…”

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Cytokinin fluoroprobe and receptor CRE1/AHK4 localize to both plasma membrane and endoplasmic reticulum

Kubiasová

Montesinos²,

Šamajová

et al. 2019

Preprint

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30 31 32 33 The plant hormone cytokinin regulates various cell and developmental processes, 34 including cell division and differentiation, embryogenesis, activity of shoot and root 35 apical meristems, formation of shoot and root lateral organs and others 1 . Cytokinins 36 are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-37 component phosphorelay cascade activate transcriptional responses in the nucleus. 38 Based on the subcellular localization of cytokinin receptors in various transient 39 expression systems, such as tobacco leaf epidermal cells, and membrane fractionation 40 experiments of Arabidopsis and maize, the endoplasmic reticulum (ER) membrane has 41 been proposed as a principal hormone perception site 2-4 . Intriguingly, recent study of 42 the cytokinin transporter PUP14 has pointed out that the plasma membrane (PM)-43 mediated signalling might play an important role in establishment of cytokinin response 44 gradients in various plant organs 5 . However, localization of cytokinin HK receptors to 45 the PM, although initially suggested 6 , remains ambiguous. Here, by monitoring 46 subcellular localizations of the fluorescently labelled natural cytokinin probe iP-NBD 7 47 and the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) 48 fused to GFP reporter, we show that pools of the ER-located cytokinin fluoroprobes and 49 receptors can enter the secretory pathway and reach the PM. We demonstrate that in 50 cells of the root apical meristem, CRE1/AHK4 localizes to the PM and the cell plate of 51 dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling 52 of the receptor and its accumulation in BFA compartments. Our results provide a new 53 perspective on cytokinin signalling and the possibility of multiple sites of perception at 54 PM and ER, which may determine specific outputs of cytokinin signalling. 55 56Fluorescently labelled analogues of phytohormones including auxin, gibberellin, 57 brassinosteroid and strigolactone have been successfully used to map the intracellular fate of 58 their receptors in planta 8 . To adopt this tool for mapping subcellular localization of cytokinin 59 receptors, using docking experiments and cytokinin activity screening bioassays, we selected 60 a fluorescently labelled bioactive compound that interacts with the binding site of a cytokinin 61 receptor. To minimize the possibility of metabolic conversion by cytokinin deactivation 62 enzymes in planta, isopentenyladenine (iP) was used as a natural cytokinin that cannot be 63 transformed through O-glycosylation at the cytokinin side chain. Furthermore, attachment of 64 7-nitro-2,1,3-benzoxadiazole (NBD), a small fluorophore, to the N9 position of iP eliminates 65 a risk of a metabolic conversion of the final cytokinin fluorescent probe iP-NBD ( Fig. 1a) 66 through N-glycosylation, or formation of cytokinin nucleotides. Docking simulations using 67 the CRE1/AHK4-iP crystal structure 9 suggested that iP-NBD may be fully embedded into the 68 active sites ...

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